In the past year we have characterized and extended our new in situ receptor-G-protein assay systems to increase their utility for a diverse family of receptor structures. We have improved the in situ reconstitution procedures to facilitate the investigation of differences in G-protein interactions for receptors expressed in mammalian cells as well as baculoviral expression in Sf9 cells. Using these techniques we have examined the bombesin receptor family (Gastrin releasing peptide receptor -- GRPr, Neuromedin B receptor -- NMBr, and bombesin receptor subtype 3, BRS3) stably expressed in mouse 3T3 fibroblasts. The GRPr and NMBr both couple exclusively to G-alpha-q and display similar selectivity for G-beta-gamma dimers. However, the two receptors differ quantitatively in Km for G-alpha-q, K1/2 for G- beta-gamma, and Vmax for catalyzed GTP exchange. The BRS3 receptor failed to couple to any of the tested G-proteins. To identify the G-protein coupling partner for BRS3, we have co- infected Sf9 cells with this receptor or GRPr together with G- alpha and G-beta-gamma viruses. In these experiments we find that the BRS3 receptor can activate, albeit partially, the mammalian G-alpha-q. Thus, we conclude that the closely related bombesin receptor structures have distinct G-protein selectivity. These data show the utility of our in situ approach for the investigation of receptor-selectivity of G-proteins. We have also extended our investigations of 5HT2c receptors by expressing the two human allelic variants of this receptor (cys and ser 23). Both of these receptors display a high basal activity, which is inhibited by classical antagonists of the 5HT2 receptor. This basal activity of the receptor is increased by high G-protein concentrations. Increasing G-protein increases the efficacy of the partial agonist mCPP to be identical to the full agonist 5HT, and at full 5HT2c-G-protein saturations the receptors are fully activated in the absence of agonists. The inverse agonist efficacy of mianserin, ketanserin and mesulergine is also increased by high G-protein saturation, so that at full saturations these antagonists inhibit receptor activity 30-fold. These studies provide a molecular insight into the activation mechanism of G-protein coupled receptors, revealing that the activated conformation of the receptor is stabilized by G-protein interaction. In situ reconstitution thus has provided a powerful approach for molecular analysis of important CNS receptor function. Lastly, we have identified the biological function of the helical domain (HD) portion of the G-protein alpha subunit revealed by the recently solved crystal structures for the retinal G-protein. We succeeded in the autonomous expression of the HD proteins from representatives of all G-protein families, and we examined the biochemical functions for the purified HD proteins using the vertebrate visual model. The HD functions as an allosteric modulator of the target enzyme for visual transduction, cyclic GMP phosphodiesterase (PDE). This HD regulation is biologically selective, only the HD proteins from G-proteins previously known to regulate the PDE displayed this activity. Further, we have characterized the molecular basis for the HD function as an entirely novel allosteric mechanism, which we suggest, may apply to all G-protein regulated enzymes.